A Multigene Assay to Predict Recurrence of Tamoxifen-Treated, Node-Negative Breast Cancer

(1)

The n e w e n g l a n d j o u r n a l of m e d i c i n e

o r i g i n a l a r t i c l e

A Multigene Assay to Predict Recurrence of

Tamoxifen-Treated, Node-Negative Breast Cancer

Soonmyung Paik, M.D., Steven Shak, M.D., Gong Tang, Ph.D., Chungyeul Kim, M.D., Joffre Baker, Ph.D., Maureen Cronin, Ph.D., Frederick L. Baehner, M.D., Michael G. Walker, Ph.D., Drew Watson, Ph.D.,

Taesung Park, Ph.D., William Hiller, H.T., Edwin R. Fisher, M.D., D. Lawrence Wickerham, M.D., John Bryant, Ph.D.,

and Norman Wolmark, M.D.

From the Division of Pathology, Operation Center, and the Biostatistics Center, Na-tional Surgical Adjuvant Breast and Bowel Project, Pittsburgh (S.P., G.T., C.K., T.P., W.H., E.R.F., D.L.W., J.B., N.W.); Genomic Health, Redwood City, Calif. (S.S., J.B., M.C., M.G.W., D.W.); the Department of Statis-tics, University of Pittsburgh, Pittsburgh (G.T., J.B.); and the University of Califor-nia, San Francisco, San Francisco (F.L.B.). Address reprint requests to Dr. Paik at the Division of Pathology, NSABP, 4 Allegheny Center, 5th Fl., East Commons Profession-al Bldg., Pittsburgh, PA 15212, or at spaik. nejm@nsabp.org.

The likelihood of distant recurrence in patients with breast cancer who have no involved lymph nodes and estrogen-receptor–positive tumors is poorly defined by clinical and histopathological measures.

m e t h o d s

We tested whether the results of a reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assay of 21 prospectively selected genes in paraffin-embedded tumor tissue would correlate with the likelihood of distant recurrence in patients with node-nega-tive, tamoxifen-treated breast cancer who were enrolled in the National Surgical Adju-vant Breast and Bowel Project clinical trial B-14. The levels of expression of 16 cancer-related genes and 5 reference genes were used in a prospectively defined algorithm to calculate a recurrence score and to determine a risk group (low, intermediate, or high) for each patient.

r e s u l t s

Adequate RT-PCR profiles were obtained in 668 of 675 tumor blocks. The proportions of patients categorized as having a low, intermediate, or high risk by the RT-PCR assay were 51, 22, and 27 percent, respectively. The Kaplan–Meier estimates of the rates of distant recurrence at 10 years in the low-risk, intermediate-risk, and high-risk groups were 6.8 percent (95 percent confidence interval, 4.0 to 9.6), 14.3 percent (95 percent confidence interval, 8.3 to 20.3), and 30.5 percent (95 percent confidence in-terval, 23.6 to 37.4). The rate in the low-risk group was significantly lower than that in the high-risk group (P<0.001). In a multivariate Cox model, the recurrence score pro-vided significant predictive power that was independent of age and tumor size (P<0.001). The recurrence score was also predictive of overall survival (P<0.001) and could be used as a continuous function to predict distant recurrence in individual pa-tients.

c o n c l u s i o n s

The recurrence score has been validated as quantifying the likelihood of distant re-currence in tamoxifen-treated patients with node-negative, estrogen-receptor–positive breast cancer.

(2)

ver the past two decades, themo

-lecular dissection of cancer has increased our understanding of the pathways that are altered in neoplastic cells.1,2

Nevertheless, the diagnosis of cancer and decisions about its treat-ment still rely largely on classic histopathological and immunohistochemical techniques. A more quantitative approach to diagnosis and rational in-dividualization of treatment are needed.

Large clinical trials, such as National Surgical Adjuvant Breast and Bowel Project (NSABP) trials B-14 and B-20, have demonstrated the benefit of tamoxifen and chemotherapy in women who have node-negative, estrogen-receptor–positive breast cancer.3-5

However, since the likelihood of distant recurrence in patients treated with tamoxifen alone after surgery is about 15 percent at 10 years, at least 85 percent of patients would be overtreated with chemotherapy if it were offered to everyone. Numer-ous attempts have been made to identify biomark-ers of residual risk,6-9

but none of them have been

recommended for guiding treatment.10-15

Molecu-lar signatures of gene expression in tumor tissue that correlate with recurrence of breast cancer have been identified by methods based on the use of DNA arrays.16-21

However, the requirement for fresh or snap-frozen tissue and uncertainties about the re-producibility of such methods have limited their clinical application.

We used a multistep approach to develop an as-say of the expression of tumor-related genes for use with routinely prepared tumor blocks and to val-idate the assay clinically. First, a high-throughput, real-time, reverse-transcriptase–polymerase-chain-reaction (RT-PCR) method was developed to quanti-fy gene expression with the use of sections of fixed,

paraffin-embedded tumor tissue.22

Second, we se-lected 250 candidate genes from the published lit-erature, genomic databases, and experiments based on DNA arrays performed on fresh-frozen tis-sue.17-19,23

Third, we analyzed data from three inde-pendent clinical studies of breast cancer involving a total of 447 patients, including the tamoxifen-only group of NSABP trial B-20, to test the relation be-tween expression of the 250 candidate genes and the recurrence of breast cancer.24-26

Fourth, we used the results of the three studies to select a panel of 16 cancer-related genes and 5 reference genes and de-signed an algorithm, based on the levels of expres-sion of these genes, to compute a recurrence score for each tumor sample. The study reported here was performed to validate the ability of the

prospective-ly defined, 21-gene RT-PCR assay and recurrence-score algorithm to quantify the likelihood of distant recurrence in patients with node-negative, estrogen-receptor–positive breast cancer who had been treat-ed with tamoxifen in the large, multicenter NSABP trial B-14.

p a t i e n t s

NSABP trial B-14 (entitled “A Clinical Trial to Assess Tamoxifen in Patients with Primary Breast Cancer and Negative Axillary Nodes Whose Tumors Are Positive for Estrogen Receptors”) enrolled 2892 pa-tients who were randomly assigned to receive place-bo or tamoxifen between January 4, 1982, and Janu-ary 25, 1988, and enrolled 1235 additional patients, all treated with tamoxifen, between January 26, 1988, and October 17, 1988. The current study of the recurrence score was approved by the Essex In-stitutional Review Board (Lebanon, N.J.) and by the institutional review boards of Allegheny General Hospital and the University of Pittsburgh (both in Pittsburgh). The need for additional informed con-sent was waived by the institutional review boards.

s a m p l e p r e p a r a t i o n

Paraffin blocks with cancer cells occupying less than 5 percent of the section area were excluded from the study. Macrodissection was performed with the use of a safety blade for cases involving nontumor elements that were amenable to macro-dissection and that constituted more than 50 per-cent of the overall area of the tissue section. RNA was extracted from three 10-µm sections when mac-rodissection had not been performed or from six 10-µm sections when macrodissection had been performed.

a s s a y m e t h o d s , g e n e s e l e c t i o n , a n d r e c u r r e n c e - s c o r e a l g o r i t h m

Gene expression in fixed, paraffin-embedded tu-mor tissue was measured as described by Cronin et al.22

The Oncotype DX assay (Genomic Health) was used. In brief, after RNA extraction and DNase I treatment, total RNA content was measured and the absence of DNA contamination was verified (as described in the Supplementary Appendix, available with the full text of this article at www.nejm.org). Reverse transcription was performed and was fol-lowed by quantitative TaqMan RT-PCR reactions in 384-well plates, performed with the use of Prism

o

(3)

m u l t i g e n e a s s a y t o p r e d i c t r e c u r r e n c e o f b r e a s t c a n c e r

7900HT instruments (Applied Biosystems). The expression of each gene was measured in triplicate and then normalized relative to a set of five

refer-ence genes (ACTB [the gene encoding b-actin],

GAPDH, GUS, RPLPO, and TFRC). Reference-nor-malized expression measurements ranged from 0 to 15, where a 1-unit increase reflected an approx-imate doubling of RNA.

The list of 21 genes and the recurrence-score algorithm (Fig. 1) were designed by analyzing the results of the three independent preliminary studies involving 447 patients and 250 candidate genes24-26 (as described in the Supplementary Appendix). The selection of the final 16 cancer-related genes was based primarily on the strength of their performance in all three studies and the consistency of primer or probe performance in the assay. The range of pos-sible recurrence scores was 0 to 100 (where higher scores indicated a greater likelihood of recurrence) and was derived from the reference-normalized ex-pression measurements for the 16 cancer-related genes.

Cutoff points were prespecified to classify pa-tients into the following categories: low risk currence score, less than 18), intermediate risk (re-currence score, 18 or higher but less than 31), and high risk (recurrence score, 31 or higher). The cut-off points were chosen on the basis of the results of NSABP trial B-20.

Reproducibility within and between blocks was assessed by performing the 21-gene assay in five serial sections from six blocks in two patients. The within-block standard deviation for the recurrence score was 0.72 recurrence-score unit (95 percent confidence interval, 0.55 to 1.04). The total within-patient standard deviation (including between-block and within-between-block standard deviations) was 2.2 recurrence-score units.

s t u d y d e s i g n a n d e n d p o i n t s

Patients were eligible if they had been randomly assigned to receive tamoxifen or had received ta-moxifen as a member of the registration group of NSABP trial B-14 and if a tumor block was available in the NSABP Tissue Bank. Exclusion criteria were insufficient tumor tissue (less than 5 percent of the overall tissue sample) as assessed by histopatho-logical analysis, insufficient RNA (less than 0.5 µg), or a weak RT-PCR signal (average cycle threshold for the reference genes, greater than 35).

The first prespecified primary objective was to determine whether the proportion of patients who

were free of a distant recurrence for more than 10 years after surgery was significantly greater in the low-risk group than in the high-risk group. The second prespecified primary objective was to deter-mine whether there was a statistically significant relation between the recurrence score and the risk of distant recurrence — one that went beyond the relation between recurrence and the standard mea-sures of the patient’s age and the size of the tumor. Contralateral disease, other second primary can-cers, and death before distant recurrence were con-sidered censoring events. Recurrence in the ipsilat-eral breast, local recurrence, and regional recurrence were not considered events or censoring events.

Figure 1. Panel of 21 Genes and the Recurrence-Score Algorithm.

The recurrence score on a scale from 0 to 100 is derived from the reference-normalized expression measurements in four steps. First, expression for each gene is normalized relative to the expression of the five reference genes (ACTB [the gene encoding b-actin], GAPDH, GUS, RPLPO, and TFRC). Reference-nor-malized expression measurements range from 0 to 15, with a 1-unit increase reflecting approximately a doubling of RNA. Genes are grouped on the basis of function, correlated expression, or both. Second, the GRB7, ER, proliferation, and invasion group scores are calculated from individual gene-expression measurements, as follows: GRB7 group score = 0.9 ¬ GRB7+0.1¬HER2 (if the result is less than 8, then the GRB7 group score is considered 8); ER group score = (0.8¬ER+1.2¬PGR+BCL2+SCUBE2)÷4; proliferation group score

=(Survivin+KI67+MYBL2+CCNB1 [the gene encoding cyclin B1]+STK15)÷5

(if the result is less than 6.5, then the proliferation group score is considered 6.5); and invasion group score=(CTSL2 [the gene encoding cathepsin L2]

+MMP11 [the gene encoding stromolysin 3])÷2. The unscaled recurrence

score (RS_U) is calculated with the use of coefficients that are predefined on the basis of regression analysis of gene expression and recurrence in the three training studies24-26

: RS_U=+0.47¬GRB7 group score¡0.34¬ER group score +1.04¬proliferation group score+0.10¬invasion group score+0.05¬CD68 ¡0.08¬GSTM1¡0.07¬BAG1. A plus sign indicates that increased expression is associated with an increased risk of recurrence, and a minus sign indicates that increased expression is associated with a decreased risk of recurrence. Fourth, the recurrence score (RS) is rescaled from the unscaled recurrence score, as follows: RS=0 if RS_U<0; RS=20¬(RS_U¡6.7) if 0≤RS_U≤100; and RS=100 if RS_U>100. Proliferation Ki67 STK15 Survivin CCNB1 (cyclin B1) MYBL2 Invasion MMP11 (stromolysin 3) CTSL2 (cathepsin L2) HER2 GRB7 HER2 Estrogen ER PGR BCL2 SCUBE2 Reference ACTB (b-actin) GAPDH RPLPO GUS TFRC GSTM1 CD68 BAG1

(4)

Prespecified secondary objectives included de-termination of the relapse-free interval (the time from surgery to any recurrence) over a 10-year peri-od and the 10-year overall mortality from any cause in the low-risk and high-risk groups; the degree of agreement in the assignment of tumor grade among three pathologists; and the performance of the recurrence score in the context of the interob-server variability in tumor grading.

No samples from trial B-14 were used for prior testing or training. The prospectively defined assay methods and end points were finalized in a proto-col signed on August 27, 2003. RT-PCR analysis was initiated on September 5, 2003, and RT-PCR data were transferred to the NSABP for analysis on September 29, 2003.

Estrogen- and progesterone-receptor proteins

were measured by ligand-binding assays. HER2 DNA

was measured by a fluorescence in situ hybridiza-tion assay (PathVysion, Vysis). Tumor grade was determined independently by three pathologists from the NSABP, Stanford University Medical Cen-ter, and the University of California, San Francisco, School of Medicine with use of a modification of

the Bloom–Richardson grading criteria.27

s t a t i s t i c a l a n a l y s i s

We tested the hypothesis that the proportion of patients who are free of a distant recurrence at 10 years would be significantly higher in the low-risk group (recurrence score, less than 18) than in the high-risk group (recurrence score, 31 or higher). The test statistic was derived by adjusting the dif-ference between the Kaplan–Meier estimates of the 10-year rate of distant recurrence in the two groups by the corresponding Greenwood variance estimates. A P value of less than 0.05 (two-sided) was considered to indicate a significant result. We also tested the hypothesis that there would be a signifi-cant difference between a (reduced) Cox proportion-al-hazards model for distant recurrence based only on age and clinical tumor size and a (full) propor-tional-hazards model based on age, clinical tumor size, and recurrence score. A P value of less than 0.05 (two-sided) in the likelihood-ratio test was considered to indicate a significant result. To de-fine the continuous relation between the recurrence score and the 10-year risk of distant recurrence, the data were fitted by a time-varying, piecewise, log-hazard ratio model with the recurrence score and its quadratic term included as covariates.28

The

10-year rate of distant recurrence was then estimated by a Breslow-type function.29

The NSABP designed the study, collected the clinical data, and analyzed the results. The assay was carried out by Genomic Health. The NSABP held the combined clinical and laboratory data (after the removal of identifying in-formation) and performed the data analyses. The manuscript was written by the NSABP, with input from Genomic Health.

c h a r a c t e r i s t i c s o f t h e p a t i e n t s

Paraffin blocks containing sufficient specimens of tissue involved by invasive breast cancer were avail-able from 675 of 2617 tamoxifen-treated patients in trial B-14. RT-PCR was successful in 668 of the 675 blocks. The 668 patients who corresponded to these blocks were similar in terms of age distribu-tion and the distribudistribu-tion of tumor size to the over-all group of 2617 tamoxifen-treated patients (Table 1 of the Supplementary Appendix). For the group of 668 patients whose tumor sample could be eval-uated, the Kaplan–Meier estimate for the propor-tion who had no distant recurrence 10 years after surgery was 85 percent.

r e c u r r e n c e r a t e s i n t h e l o w - r i s k a n d h i g h - r i s k g r o u p s

The Kaplan–Meier estimate for the proportion of patients in the low-risk group who were free of a distant recurrence at 10 years (93.2 percent) was sig-nificantly greater than the proportion in the high-risk category (69.5 percent) (P<0.001) (Table 1 and

r e s u l t s

* A low risk was defined as a recurrence score of less than 18, an intermediate risk as a score of 18 or higher but less than 31, and a high risk as a score of 31 or higher. † CI denotes confidence interval.

‡ P<0.001 for the comparison with the low-risk category. Table 1. Kaplan–Meier Estimates of the Rate of Distant Recurrence at 10 Years, According to Recurrence-Score Risk Categories.* Risk Category Percentage of Patients Rate of Distant Recurrence at 10 Yr (95% CI)† percent Low 51 6.8 (4.0–9.6) Intermediate 22 14.3 (8.3–20.3) High 27 30.5 (23.6–37.4)‡

(5)

Fig. 2). The recurrence score was also significantly correlated with two secondary end points: the re-lapse-free interval and overall survival (P<0.001 for both) (Fig. 2B and 2C of the Supplementary Ap-pendix).

r e c u r r e n c e s c o r e , a g e , t u m o r s i z e , a n d r i s k o f d i s t a n t r e c u r r e n c e

As expected, younger patients (those less than 50 years of age) had higher rates of distant recurrence at 10 years than older patients (21.1 percent [95 percent confidence interval, 15.1 to 26.8 percent] vs. 12.3 percent [95 percent confidence interval, 9.1 to 15.3 percent]), whereas patients with smaller tumors (diameter, 2 cm or less) had lower estimat-ed rates of distant recurrence at 10 years than those with larger tumors (13.3 percent [95 percent confi-dence interval, 9.9 to 16.8 percent] vs. 17.5 percent [95 percent confidence interval, 12.6 to 22.3 per-cent]). In a multivariate Cox model in which distant recurrence was evaluated in relation to both age and tumor size, age alone was significantly corre-lated with distant recurrence (P=0.004, with young-er patients more likely to have recurrence), whyoung-ere- where-as tumor size trended toward significance (P=0.06, with larger tumors more likely to recur) (Table 2). In a multivariate Cox model in which distant recur-rence was evaluated in relation to the recurrecur-rence score, age, and tumor size, the recurrence score pro-vided significant predictive power that was inde-pendent of age and tumor size (P<0.001) (Table 2). When recurrence score was added to the model, age and tumor size were no longer statistically sig-nificant. Similar results were observed when more than two categories of age and tumor size were used in the model (data not shown).

e s t r o g e n - a n d p r o g e s t e r o n e - r e c e p t o r p r o t e i n s a n d a m p l i f i c a t i o n o f h e r 2

No relation was observed between the levels of es-trogen- or progesterone-receptor proteins and the risk of distant recurrence (Fig. 1 of the

Supplemen-tary Appendix). HER2 was amplified in 55 of the

668 tumors (8.2 percent) and not amplified in 605 tumors (90.6 percent); the result was indetermi-nate in 8 (1.2 percent). The Kaplan–Meier estimate of the proportion of patients free of distant re-currence at 10 years among those with tumors in

which HER2 was amplified was 75.0 percent (95

percent confidence interval, 63.2 to 86.9 percent), and 86.0 percent (95 percent confidence interval,

Figure 2. Likelihood of Distant Recurrence, According to Recurrence-Score Categories.

A low risk was defined as a recurrence score of less than 18, an intermediate risk as a score of 18 or higher but less than 31, and a high risk as a score of 31 or higher. There were 28 recurrences in the low-risk group, 25 in the inter-mediate-risk group, and 56 in the high-risk group. The difference among the groups is significant (P<0.001).

Freedom from Distant Recurrence

(% of patients) 80 70 90 60 40 30 10 50 20 0 0 2 4 6 8 10 12 14 16 Years Low risk High risk Intermediate risk 100 No. at Risk Low risk Intermediate risk High risk 38 16 13 170 66 63 231 80 83 258 96 91 276 104 105 298 116 119 313 128 137 328 139 154 338 149 181

* Age at surgery was a binary variable (0 for an age of less than 50 years and 1 for an age of 50 years or more); clinical tumor size was a binary variable (0 for a diam-eter of 2 cm or less and 1 for a diamdiam-eter greater than 2 cm); and the recurrence score was a continuous variable, with the hazard ratio for distant recurrence calculated relative to an increment of 50 units (chosen to dichotomize the re-currence score and thus improve comparability of the hazard ratio with the hazard ratios based on the clinical covariates).

† CI denotes confidence interval.

‡ P<0.001 and chi-square=33.7 for the comparison with the analysis without the recurrence score (by the likelihood-ratio test).

Table 2. Multivariate Cox Proportional Analysis of Age, Tumor Size, and Recurrence Score in Relation to the Likelihood of Distant Recurrence.*

Variable P Value

Hazard Ratio (95% CI)† Analysis without recurrence score

Age at surgery 0.004 0.57 (0.39–0.83)

Clinical tumor size 0.06 1.44 (0.99–2.11)

Analysis with recurrence score‡

Age at surgery 0.08 0.71 (0.48–1.05)

Clinical tumor size 0.23 1.26 (0.86–1.86) Recurrence score <0.001 3.21 (2.23–4.61)

(6)

83.1 to 88.9 percent) among patients with tumors

in which HER2 was not amplified (P=0.08) (Fig.

2A of the Supplementary Appendix). In Cox models that included the recurrence score and traditional measures (estrogen receptor, progesterone recep-tor, or DNA amplification of HER2), only the

recur-rence score was a significant predictor of distant recurrence (data not shown).

r e c u r r e n c e s c o r e , t u m o r g r a d e , a n d r i s k o f d i s t a n t r e c u r r e n c e

The assessment of tumor grade by each of the three pathologists correlated with the risk of distant re-currence (Tables 2A, 2B, and 2C of the Supplemen-tary Appendix). The recurrence score provided sig-nificant information beyond tumor grade for each of the three pathologists (P<0.001). The concor-dance in assessment of grade between any two pa-thologists was 59 to 65 percent, and the overall con-cordance among all the three pathologists was 43 percent (Table 3 of the Supplementary Appendix). Agreement among the three pathologists was low-est for well-differentiated and moderately differ-entiated tumor grades (kappa, 0.36 and 0.23, respec-tively) and highest for a poorly differentiated grade (kappa, 0.61).

Finally, multivariate Cox proportional-hazards analyses were performed to explore the relation be-tween distant recurrence and age, tumor size, tumor

grade, HER2 amplification, amounts of

estrogen-and progesterone-receptor protein, estrogen-and recurrence score (Table 3). The recurrence score and poor tu-mor grade were significant predictors of distant re-currence.

r i s k o f d i s t a n t r e c u r r e n c e i n s u b g r o u p s o f p a t i e n t s

The recurrence score predicted distant recurrence for all age categories and all categories of tumor size (Fig. 3). Patients with a low-risk recurrence score (less than 18) had less frequent distant recur-rences at 10 years than patients with a high-risk score (31 or higher). Moreover, not all patients with small tumors (109 patients with a tumor 1 cm in di-ameter or smaller) were at low risk; the recurrence score identified 44 of those patients as having an intermediate or high risk and a 15 to 20 percent risk of distant recurrence at 10 years.

The subgroup of patients with moderately dif-ferentiated tumors (the most common grade) could be distinguished to be at low or high risk by the recurrence score (Fig. 3). A subgroup of patients with well-differentiated tumors had high recurrence scores and high rates of distant recurrence. For two of the three pathologists, a subgroup of patients with poorly differentiated tumors had low recur-rence scores and low rates of distant recurrecur-rence (Fig. 3A and 3B of the Supplementary Appendix).

* The tumor grades were those of one of the three pathologists. Age at surgery was a binary variable (0 for an age of less than 50 years and 1 for an age of 50 years or more); clinical tumor size was a binary variable (0 for a diameter of 2 cm or less and 1 for a diameter greater than 2 cm); grade was a binary variable (poorly differentiated relative to well differentiated and moderately differenti-ated relative to well differentidifferenti-ated); HER2 amplification was a binary variable (0 for no amplification on fluorescence in situ hybridization and 1 for amplifi-cation); the amount of estrogen-receptor protein was an ordinal variable, with the baseline level being 10 to 49 fmol per milligram; and recurrence score was a continuous variable, with the hazard ratio for distant recurrence calculated relative to an increment of 50 units.

† CI denotes confidence interval.

‡ P<0.001 and chi-square=15.2 for the comparison with the analysis without the recurrence score.

Table 3. Multivariate Cox Proportional Analysis of Age, Tumor Size, Tumor Grade, and Recurrence Score in Relation to the Likelihood of Distant Recurrence.*

Variable P Value

Hazard Ratio (95% CI)† Analysis without recurrence score

Age at surgery 0.10 0.70 (0.45–1.07)

Clinical tumor size 0.13 1.35 (0.92–1.98) Tumor grade Moderately differentiated 0.04 1.87 (1.04–3.37) Poorly differentiated <0.001 5.14 (2.89–9.15) HER2 amplification 0.89 1.04 (0.57–1.90) Estrogen-receptor protein 50–99 fmol/mg 0.23 0.71 (0.41–1.24) 100–199 fmol/mg 0.38 0.78 (0.45–1.35) ≥200 fmol/mg 0.90 0.97 (0.55–1.69)

Analysis with recurrence score‡

Age at surgery 0.22 0.76 (0.50–1.18)

Clinical tumor size 0.38 1.19 (0.81–1.76) Tumor grade Moderately differentiated 0.15 1.55 (0.85–2.81) Poorly differentiated <0.001 3.34 (1.79–6.26) HER2 amplification 0.06 0.51 (0.26–1.02) Estrogen-receptor protein 50–99 fmol/mg 0.32 0.75 (0.43–1.31) 100–199 fmol/mg 0.72 0.90 (0.52–1.58) ≥200 fmol/mg 0.94 1.02 (0.58–1.70) Recurrence score <0.001 2.81 (1.70–4.64)

(7)

r e c u r r e n c e s c o r e a s a c o n t i n u o u s p r e d i c t o r o f d i s t a n t r e c u r r e n c e

The likelihood of distant recurrence at 10 years increased continuously as the recurrence score in-creased (Fig. 4). Two-sided confidence intervals for the likelihood of distant recurrence are generally ±2 to 3 percent for recurrence scores of less than 30 and ±3 to 5 percent for recurrence scores of 30 to 50. For recurrence scores greater than 50, the like-lihood of distant recurrence increases only slightly as the score increases. On average, patients with re-currence scores greater than 50 (12 percent of the 668 patients) had a risk of distant recurrence at 10 years of 33.8 percent (95 percent confidence inter-val, 23.4 to 44.2 percent).

Using a prospectively defined gene-expression assay and an algorithm for calculating recurrence scores, we were able to quantify the likelihood of distant recurrence in patients with node-negative, estrogen-receptor–positive breast cancer who had been treated with tamoxifen. The difference in the risk of distant recurrence between patients with low recurrence scores and those with high recurrence scores was large and statistically significant. Many patients (51 percent of the patients in the study) were categorized as having a low risk, and their rate of distant recurrence at 10 years was 6.8 percent. A smaller group of patients (27 percent) was cate-gorized as having a high risk; their rate of distant recurrence at 10 years was 30.5 percent — a risk similar to that observed among patients with node-positive disease.30

The use of the recurrence score as a continuous predictor provides an accurate esti-mate of the risk of distant recurrence in individual patients.

The recurrence score can also predict overall

sur-d i s c u s s i o n

20 40 60 80 90

10 30 50 70 100

Percentage of Patients Free of Distant Recurrence at 10 Yr All patients Subgroup No. of Patients Age (yr) Tumor size (cm) <40 Low risk Intermediate risk High risk 40–50 All patients Low risk Intermediate risk High risk 50–60 All patients Low risk Intermediate risk High risk >60 All patients Low risk Intermediate risk High risk Tumor grade Well differentiated All patients Low risk Intermediate risk High risk Moderately differentiated All patients Low risk Intermediate risk High risk Poorly differentiated All patients Low risk Intermediate risk High risk 1.1–2.0 All patients Low risk Intermediate risk High risk 2.1–4.0 All patients Low risk Intermediate risk High risk >4.0 All patients Low risk Intermediate risk High risk ≤1.0 All patients Low risk Intermediate risk High risk 668 59 16 10 33 135 66 29 40 173 81 48 44 301 175 62 64 224 166 41 17 296 139 80 77 148 33 28 87 305 149 72 84 220 110 44 66 34 14 6 14 109 65 27 17 All patients

Figure 3. Kaplan–Meier Estimates of the Proportion of Patients Free of Distant Recurrences at 10 Years, According to Age, Tumor Size, and Tumor Grade.

For each group of patients, the results for low-, interme-diate-, and high-risk recurrence-score categories (scores of less than 18, 18 or higher but less than 31, and 31 or higher, respectively) are shown. The tumor grades are those of one of the three pathologists. The size of each square corresponds to the size of the subgroup; the hor-izontal lines represent the 95 percent confidence interval.

(8)

vival. This feature is notable, since approximately 50 percent of the deaths occurred in the absence of recurrent breast cancer. In addition, the recurrence score predicts the relapse-free interval (including the interval free of local and regional recurrences). Thus, the recurrence score correlates in a statisti-cally significant manner with all the end points we examined.

The patient’s age and the size of the tumor are routinely used as predictors of recurrence in breast cancer and are incorporated into current treatment guidelines.13-15

When the recurrence score was combined with data pertaining to age and tumor size to predict the risk of distant recurrence, only the recurrence score remained statistically signif-icant in a multivariate analysis. It is likely that the decreased risk of recurrence in older patients is not related to age itself but instead, at least in part, to the higher amount of estrogen-receptor protein in older patients’ tumors.31,32

The contribution of

ER expression to the recurrence score captures

this factor.

The subgroup analysis of patients according to age and tumor size was exploratory, and the results should be interpreted cautiously. Nevertheless, the recurrence score was a consistent predictor of dis-tant recurrence in patients of all age categories and

all tumor-size categories. For example, more than a third of the patients with small tumors (1 cm in di-ameter or smaller) had intermediate-risk or high-risk recurrence scores and a 15 to 20 percent high-risk of distant recurrence.

We evaluated the recurrence score in the context of the interobserver variability in tumor grading that is typical in oncology practice. Tumor grade cor-relates with the likelihood of recurrence when an-alyzed in large populations of patients. However, previous studies have also documented that the grading of breast cancer entails a degree of subjec-tive judgment, leading to low concordance among pathologists. Robbins et al.33

compared the inter-observer reproducibility in their study to the pub-lished results of four other groups.34-36

Complete agreement in those five studies ranged from 54 per-cent to 83 perper-cent (kappa, 0.17 to 0.73). We found that the concordance among pathologists for the poorly differentiated grade is moderate (kappa, 0.61) and for the well-differentiated and moderately differentiated grades is low (kappa, 0.23 and 0.36, respectively). Recently, a Breast Task Force serv-ing the American Joint Committee on Cancer did not add tumor grade to its staging criteria because of the sparseness and variability of the data.37

Traditional measures of estrogen-receptor

pro-tein (by ligand-binding assay) and HER2 (by

fluo-rescence in situ hybridization) in this study were only weakly predictive of the risk of distant recur-rence. The quantitative information that the RT-PCR assay provides for ER, HER2, and the other 14 can-cer-related genes is clearly important.

It is important to emphasize that we do not know whether the genes used in the calculation of the recurrence score correlate with recurrence in the population we studied because they show a relation with the natural history of breast cancer, because they predict responsiveness to tamoxifen, or both. Esteva et al.38

found no correlation between the re-currence score and the rate of distant rere-currence in 149 selected patients with node-negative breast can-cer who did not receive adjuvant systemic therapy. However, in that cohort, patients with well-differ-entiated tumors (i.e., those with a low nuclear grade) had a surprisingly worse survival rate than patients with moderately differentiated or poorly differenti-ated tumors. The current data cannot be used to se-lect women for tamoxifen therapy.

Few assays have been rigorously validated for use as prognostic or predictive tests in oncology. We conducted a prospectively designed validation Figure 4. Rate of Distant Recurrence as a Continuous Function of the

Recur-rence Score.

The continuous function was generated with use of a piecewise log-hazard-ratio model.28

The dashed curves indicate the 95 percent confidence interval. The rug plot on top of the x axis shows the recurrence score for individual pa-tients in the study.

Rate of Distant Recurrence at 10 Yr (% of patients)

30 35 25 20 10 5 15 0 0 5 10 15 20 25 30 35 40 45 50 Recurrence Score Low-Risk Group

Intermediate-Risk Group High-Risk Group

(9)

study of a multigene-expression assay in a large, multicenter clinical trial. It is of practical impor-tance that this assay involves the use of very small amounts of the tumor tissue that is routinely pre-pared after surgery.

Supported by the National Surgical Adjuvant Breast and Bowel Project and Genomic Health. Genomic Health paid the costs of shipping the paraffin-embedded tissue sections and performing all RT-PCR assays.

Drs. Paik, Shak, Baker, Cronin, Walker, and Bryant report hold-ing a patent for the RT-PCR assay used in this study. Drs. Shak, Baker, Cronin, and Watson report holding equity ownership or stock

op-tions in Genomic Health and being employed by Genomic Health, the commercial entity that sponsored the study. Dr. Walker reports having received consulting fees from Genomic Health and owning stock options. Dr. Baehner reports having received consulting fees from Genomic Health; Dr. Paik, lecture fees from Genomic Health; and Dr. Wickerham, consulting fees from AstraZeneca.

We are indebted to Tracy George (Stanford University); to Terry Mamounas (NSABP) for his comments; to Randy Scott, Debjani Dutta, Daniel Klaus, Mylan Pho, Anhthu Nguyen, Jennie Jeong, Stephanie Butler, Joel Robertson, Ken Stineman, Marti Haskins, and Claire Alexander (all of Genomic Health); and to Clifford Hudis, Tom Fleming, David Botstein, David Agus, and Fred Cohen for their helpful advice and suggestions.

r e f e r e n c e s

1. Buetow KH, Klausner RD, Fine H, Kap-lan R, Singer DS, Strausberg RL. Cancer Molecular Analysis Project: weaving a rich cancer research tapestry. Cancer Cell 2002; 1:315-8.

2. Hahn WC, Weinberg RA. Rules for mak-ing human tumor cells. N Engl J Med 2002; 347:1593-603. [Erratum, N Engl J Med 2003; 348:674.]

3. Fisher B, Costantino J, Redmond C, et al. A randomized clinical trial evaluating ta-moxifen in the treatment of patients with node-negative breast cancer who have estro-gen-receptor-positive tumors. N Engl J Med 1989;320:479-84.

4. Fisher B, Jeong JH, Bryant J, et al. Treat-ment of lymph-node-negative, oestrogen-receptor-positive breast cancer: long-term findings from National Surgical Adjuvant Breast and Bowel Project randomised clini-cal trials. Lancet 2004;364:858-68.

5. Fisher B, Dignam J, Wolmark N, et al. Tamoxifen and chemotherapy for lymph node-negative, estrogen receptor-positive breast cancer. J Natl Cancer Inst 1997;89: 1673-82.

6. Bryant J, Fisher B, Gunduz N, Costanti-no JP, Emir B. S-phase fraction combined with other patient and tumor characteristics for the prognosis of node-negative, estrogen-receptor-positive breast cancer. Breast Can-cer Res Treat 1998;51:239-53.

7. Henderson IC, Patek AJ. The relation-ship between prognostic and predictive fac-tors in the management of breast cancer. Breast Cancer Res Treat 1998;52:261-88.

8. Hayes DF. Do we need prognostic factors in nodal-negative breast cancer? Arbiter. Eur J Cancer 2000;36:302-6.

9. Esteva FJ, Hortobagyi GN. Prognostic molecular markers in early breast cancer. Breast Cancer Res 2004;6:109-18.

10.Fitzgibbons PL, Page DL, Weaver D, et al. Prognostic factors in breast cancer: College of American Pathologists Consensus State-ment 1999. Arch Pathol Lab Med 2000;124: 966-78.

11.Clinical practice guidelines for the use of tumor markers in breast and colorectal can-cer: adopted on May 17, 1996 by the Ameri-can Society of Clinical Oncology. J Clin Oncol 1996;14:2843-77.

12.Bast RC Jr, Ravdin P, Hayes DF, et al. 2000 Update of recommendations for the use of tumor markers in breast and colorec-tal cancer: clinical practice guidelines of the American Society of Clinical Oncology. J Clin Oncol 2001;19:1865-78. [Errata, J Clin Oncol 2001;19:4185-8, 2002;20:2213.]

13.Goldhirsch A, Glick JH, Gelber RD, Coates AS, Senn HJ. Meeting highlights: International Consensus Panel on the Treat-ment of Primary Breast Cancer: Seventh In-ternational Conference on Adjuvant Thera-py of Primary Breast Cancer. J Clin Oncol 2001;19:3817-27.

14.Eifel P, Axelson JA, Costa J, et al. National Institutes of Health Consensus Development Conference Statement: adjuvant therapy for breast cancer, November 1-3, 2000. J Natl Cancer Inst 2001;93:979-89.

15.Carlson RW, Edge SB, Theriault RL. NCCN: breast cancer. Cancer Control 2001; 8:Suppl 2:54-61.

16.Davis RE, Staudt LM. Molecular diag-nosis of lymphoid malignancies by gene expression profiling. Curr Opin Hematol 2002;9:333-8.

17.Perou CM, Sorlie T, Eisen MB, et al. Mol-ecular portraits of human breast tumours. Nature 2000;406:747-52.

18.Golub TR, Slonim DK, Tamayo P, et al. Molecular classification of cancer: class dis-covery and class prediction by gene expres-sion monitoring. Science 1999;286:531-7.

19.van ’t Veer LJ, Dai H, van de Vijver MJ, et al. Gene expression profiling predicts clini-cal outcome of breast cancer. Nature 2002; 415:530-6.

20.van de Vijver MJ, He YD, van ’t Veer LJ, et al. A gene-expression signature as a pre-dictor of survival in breast cancer. N Engl J Med 2002;347:1999-2009.

21.Schena M, Shalon D, Davis RW, Brown PO. Quantitative monitoring of gene expres-sion patterns with a complementary DNA microarray. Science 1995;270:467-70.

22.Cronin M, Pho M, Dutta D, et al. Mea-surement of gene expression in archival par-affin-embedded tissues: development and performance of a 92-gene reverse transcrip-tase-polymerase chain reaction assay. Am J Pathol 2004;164:35-42.

23.Sorlie T, Perou CM, Tibshirani R, et al.

Gene expression patterns of breast carcino-mas distinguish tumor subclasses with clin-ical implications. Proc Natl Acad Sci U S A 2001;98:10869-74.

24. Esteban J, Baker J, Cronin M, et al. Tumor gene expression and prognosis in breast cancer: multi-gene RT-PCR assay of paraf-fin-embedded tissue. Prog Proc Am Soc Clin Oncol 2003;22:850. abstract.

25.Cobleigh MA, Bitterman P, Baker J, et al. Tumor gene expression predicts distant dis-ease-free survival (DDFS) in breast cancer patients with 10 or more positive nodes: high throughout RT-PCR assay of paraffin-embed-ded tumor tissues. Prog Proc Am Soc Clin Oncol 2003;22:850. abstract.

26.Paik S, Shak S, Tang G, et al. Multi-gene RT-PCR assay for predicting recurrence in node negative breast cancer patients — NSABP studies B-20 and B-14. Breast Cancer Res Treat 2003;82:A16. abstract.

27.Elston CW, Ellis IO. Pathological prog-nostic factors in breast cancer. I. The value of histological grade in breast cancer: experi-ence from a large study with long-term fol-low-up. Histopathology 1991;19:403-10.

28.Gray RJ. Flexible methods for analyzing survival data using splines, with applications to breast cancer prognosis. J Am Stat Assoc 1992;87:942-51.

29.Valenta Z, Weissfeld L. Estimation of the survival function for Gray’s piecewise-con-stant time-varying coefficients model. Stat Med 2002;21:717-27.

30.Fisher B, Redmond C, Legault-Poisson S, et al. Postoperative chemotherapy and tamoxifen compared with tamoxifen alone in the treatment of positive-node breast can-cer patients aged 50 years and older with tu-mors responsive to tamoxifen: results from the National Surgical Adjuvant Breast and Bowel Project B-16. J Clin Oncol 1990;8: 1005-18.

31.Fisher B, Wickerham DL, Brown A, Red-mond CK. Breast cancer estrogen and pro-gesterone receptor values: their distribution, degree of concordance, and relation to num-ber of positive axillary nodes. J Clin Oncol 1983;1:349-58.

32.Anderson WF, Chatterjee N, Ershler WB, Brawley OW. Estrogen receptor breast cancer phenotypes in the Surveillance,

(10)

Epi-m u l t i g e n e a s s a y t o p r e d i c t r e c u r r e n c e o f b r e a s t c a n c e r

demiology, and End Results database. Breast Cancer Res Treat 2002;76:27-36.

33.Robbins P, Pinder S, de Klerk N, et al. Histological grading of breast carcinomas: a study of interobserver agreement. Hum Pathol 1995;26:873-9.

34.Hopton DS, Thorogood J, Clayden AD, MacKinnon D. Observer variation in histo-logical grading of breast cancer. Eur J Surg Oncol 1989;15:21-3.

35.Davis BW, Gelber RD, Goldhirsch A, et

al. Prognostic significance of tumor grade in clinical trials of adjuvant therapy for breast cancer with axillary lymph node metastasis. Cancer 1986;58:2662-70.

36.Theissig F, Kunze KD, Haroske G, Meyer W. Histological grading of breast cancer: interobserver, reproducibility and prognos-tic significance. Pathol Res Pract 1990;186: 732-6.

37.Singletary SE, Allred C, Ashley P, et al. Revision of the American Joint Committee

on Cancer staging system for breast cancer. J Clin Oncol 2002;20:3628-36.

38. Esteva FJ, Sahin AA, Coombes K, et al. Multi-gene RT-PCR assay for predicting re-currence in node negative breast cancer pa-tients — MD Anderson Clinical Validation Study. Breast Cancer Res Treat 2003;82:A16. abstract.